Biochemical conversions by yeast fermentation at high cell densities

ABSTRACT

A method of high cellular density yeast fermentation at high mineral salts feed to and maintained in the ferment. 
     Single cell protein (SCP) is produced in an aerobic fermentation process at high yields under high cell density conditions employing media of high mineral salts concentration. Novel Pichia pastoris and Hansenula polymorpha yeasts are disclosed.

This application is a continuation-in-part of application Ser. No.110,457 filed Jan. 15, 1980, now abandoned, which is acontinuation-in-part of application Ser. No. 29,418 filed Apr. 12, 1979,now abandoned.

FIELD OF THE INVENTION

The invention pertains to biochemical conversions. In one aspect, theinvention relates to single cell protein. In another aspect, theinvention pertains to the production of single cell protein. In afurther aspect, the invention pertains to novel yeast strains. In yetanother aspect, the invention pertains to chemical conversions bymicrobiological action.

BACKGROUND OF THE INVENTION

Efforts to relieve world-wide shortages of protein have included variousbio-synthesis processes in which single cell protein (SCP) is obtainedby the growth of one or another of a variety of microogranisms onvarious carbon-containing substrates.

The carbon energy substrates should be readily available, relativelycheap, uniform, and safe. Petroleum hydrocarbons have been employed ascarbon energy source, but have faced practical difficulties in the lackof water-solubility and in the high consumption of molecular oxygenneeded to assist in the microbial conversion. Other processes havecentered on the use of oxygenated hydrocarbon derivatives as feedstocksdue to their relative water-solubility and hence ease of handling in anaqueous ferment, and in the substantially reduced molecular oxygenrequirements for the microbial conversion-growth process.

However, a limiting factor in the commercialization of single cellprotein processes has been the necessity to control the ferment atrelatively moderate cell densities, with but moderate yields of driedcells based on substrate consumed, and the consequent necessity tohandle large amounts of total fermentation effluent liquor in order torecover the moderate amounts of SCP material. Handling large quantitiesof aqueous fermentation effluent liquor complicates concentration of thesingle cell protein product in such as centrifuges, as well as washingand drying steps.

Some processes in the past have concentrated on the culturing ofbacteria because of the slightly higher crude protein contents of thecell as compared to the content obtainable from yeast in general.However, yeasts are widely available and relatively simply cultured.Yeast cells generally are slightly larger as compared to bacteria cells,and, hence, yeast cells tend to be more easily separated from thefermentation effluent liquor.

Discovery of means and methods to increase cell yields, and particularlyto operate and maintain continuous production at high cell densities,would be highly desirable. The resultant handling of substantially lessfermentation liquor effluent volume, for example, would mean largesavings in reduced sizes of piping and pumps, reduced makeup waterrequirements with reduced sterilization requirements, and reducedrequirements of equipment sizing and handling for coagulation andseparation processes.

Growth of yeast cells at high cell densities further allows moreefficient assimilation of substrate in a smaller fermentation apparatus,useful, for example, in an effluent scrubbing scheme such as disclosedin U.S. Pat. No. 3,646,239. Yeast cells grown at high cell densitiesproduce extracellular products in excellent yields, useful, for example,in an enhanced oil recovery process such as disclosed in U.S. Pat. No.4,261,420, wherein a high cell density fermentation process will providemaximum CO₂ production for oil recovery purposes. Other biochemicalconversions using yeasts which can benefit from the high cell densityfermentation process of my invention include the oxidation of alkanes todicarboxylic acids (U.S. Pat. No. 3,796,630), the oxidation of C₁₄ toC₁₈ 1-alkanes to epoxides and glycols Fonken, G. S., and R. a. Johnson,Chemical Oxidations With Microorganisms (Dekker, N.Y., 1972) pp 113-115,the oxidation of alcohols to ketones (U.S. Pat. No 4,250,259, U.S. Pat.Nos. 4,268,630 and 4,269,940), and the production of extracellularbiopolymers for use as viscosifying agents in aqueous media, e.g. in oilfield water flooding applications (U.S. Pat. No. 3,312,279); thedisclosures of all of these patents/articles being herein incorporatedin total by reference, since all are amenable to improvement by my highsalts feed/high cellular density fermentation method.

SUMMARY OF THE INVENTION

I have discovered a method which enables yeast-fermentations to beconducted at very high cellular densities, continuously, by a high saltsfeed method. My method of imposing a high salts force feed to theferment results in a high cellular density of yeast cells contained inthe ferment. My high salts/high cell density method is effective, andmaintains high cell densities without the heretofore art taughtnecessity of physical means of cellular build-up by either removingferment and separating cells for recycle to the fermentor, or to use aninternal separator in the fermentor in order to separate out liquor forremoval and thus build up cell counts, either method being expensive,difficult, at times harmful to the cells, lacking adequate ease ofcontrol, and add markedly to the apparatus costs.

My method of high cell density fermentation results in high rate usageof substrate, high (bio) chemical conversions, and desirable levels ofextracellular products production.

I have discovered a way to operate a continuous aerobic fermentationprocess employing yeast cultures so as to enable operation of theferment in the fermentor at very high cell densities ordinarilyunobtainable. My method achieves high levels of substrate utilization byadding media containing high mineral salts concentrations to the fermentin the fermentor. This enables high yields of yeast single cell proteinand for high levels of extracellular product formation.

Cell density is defined as the concentration of cells by weight on a drybasis per volume of ferment. The ferment is defined as the total volumeof aqueous fermentation broth or liquor, including cells. Cell densityusually is expressed as grams/liter. Yield is defined as the amount ofcells by weight produced for a given consumption by weight of carbonenergy source or substrate fed to the ferment, and is usually expressedin grams/gram.

The high cell densities obtainable by my process significantlystreamline and reduce the cost of single cell protein production, and ofchemical conversion promoted by yeasts. Concentrating the resultingsingle cells as protein product by such as centrifuge means in manyinstances is sharply reduced or even eliminated. The cellular product(cells) can be, if desired, washed in washing means to remove residualunconsumed salts and extra-cellular products such as amino acids,biopolymers extracellular enzymes, and the like. The washed cells canthen be sent directly to a dryer means such as a spray dryer. Thewashings contain most of the remaining mineral salts not incorporatedinto the cells and the extracellular products. The washings can betreated to recover or isolate extracellular products such as thebiopolymers, enzymes, etc., and the unconsumed salts can be recycled tothe fermentor as may be appropriate.

Requirements for water to the fermentation step thus are reducedconsiderably, and, importantly, there is little or no waste waterrequiring disposal. Alternatively, if desired, the total fermentincluding residual salts and extracellular products can be dried.

Heretofore, continuous processes for the production of single cellprotein materials from yeast cultures typically gave ferment effluentsof relatively low yeast cell contents, such as about 20 to 25 grams ofcelles per liter of ferment. My invention, however, provides a processwhereby the fermentation step produces yeast cells at relatively veryhigh cell density levels, such as above 100 grams per liter of ferment.Such high cell densities in the ferment, coupled with high yields ofyeast cells, mean more efficient effective production of yeast cells andextracellular products, as well as more efficient utilization ofsubstrate, particularly under continuous production conditions.

HIGH SALTS/HIGH CELL DENSITY APPLICATIONS

Hereinabove, I have described, with regard to my high salts/high celldensity fermentation invention, various applications of my invention toboth production of single cells to obtain cellular protein and enzymesas well as various extracellular products, and to various biochemicalconversions. Where the biochemical conversion is the prime objective,the coproduced cells may in a sense be incidental or a by-product.

My invention is applicable to any process using appropriate yeasts in anaqueous aerobic fermentation process to achieve, by the yeasts or theirenzymes, a biochemical conversion. These include the oxidation to carbondioxide of a variety of substrates such as the alcohols; biomass toalcohol; oxygenated hydrocarbons to biopolymers suitable forviscosifiers or drilling fluids; production of amino acids; ofphosphomannan gums (phosphorylated mannan) from glucose (See U.S. Pat.No. 3,312,279); biocellulose; conversion of hydrocarbons to alcohols,ketones and/or organic acids; alkenes to glycols and epoxides; alcoholsto ketones; production of variety of medicines as extracellularproducts; and others. Even old yeast fermentation processes such asconversion of sugars to alcohols, or to acids can be conducted moreefficiently in smaller equipment by my high salts/high cell densitymethod. Refer for example to Oura, Process Biochemistry, April 1977, pp19-21 and 35; and Perlman, Chemtech, April 1974, pp 210-215.

DETAILED DISCLOSURE OF THE INVENTION

In accordance with my invention, yeasts are grown under substantiallycontinuous aerobic aqueous fermentation conditions on a suitable carbonenergy source as substrate, employing effective amounts of molecularoxygen-containing gases, assimilable-nitrogen source, high input ofnitrient mineral salts, and, where necessary, adding additional nutrientorganic material such as vitamins such as biotin and/or thiamine.

In my process the mineral salts are added at high levels as will bedescribed hereinafter, resulting in a continuous fermentation processoperating at high cell densities, with high yields.

My invention essentially lies in almost the force-feeding of the cellsby adding highly concentrated (relatively) mineral salts into theferment, resulting in high growth rates of the yeast. The mineral saltsconcentration in the liquid supernatant of the ferment (that is, theferment excluding cells) itself remains, of course, at relatively lowlevels, since the salts are consumed by the yeast cells in growth andreproduction. The salts concentration in the cells plus liquidsupernatant, thus, is very high in my process.

FERMENTATION CONDITIONS

Culturing is accomplished in a growth medium comprising an aqueousmineral salts medium, the carbon energy source material, molecularoxygen, assimilable nitrogen, and of course, a starting inoculum of oneor more particular species of yeast microorganisms to be employed.

In my invention, high concentrations of mineral salts are fed to theferment and high concentrations are maintained in the ferment. It isnecessary to supply suitable amounts in proper proportions of selectedmineral nutrients in the feed media, in order to assure propermicroorganism growth, to maximize assimilation of the carbon and energysource by the cells in the microbial conversion process, and to achievemaximum cellular yields with maximum cell density in the fermentationmedia.

Although the composition of the ferment can vary over a wide range,depending in part on the yeast and substrate employed, the mineralscontent in the ferment (that is, liquid plus cells) in accordance withmy invention is relatively high, at higher levels than heretoforeconsidered suitable or practiced by the prior art. Set forth in thetable below are the minimum, broad, and presently preferred ranges ofconcentrations of various elements in the ferment, the concentrationbeing expressed as of the element, though it is recognized that all orpart of each can be present in the form of a soluble ion, or in casessuch as P are present in a combined form of some type such as phosphate.The amount of each element is expressed in grams or milligrams per literof ferment (aqueous phase, including cells):

                  TABLE I                                                         ______________________________________                                        Weight of Element per Liter of Ferment                                        Element Minimum   Broad Range Preferred Range                                 ______________________________________                                        P       1.9 g     2.9-20 g    2.2-10 g                                        K       1 g       1-20 g      1.5-10 g                                        Mg      0.15 g    0.15-3 g    0.3-1.2 g                                       Ca      0.06 g    0.06-1.6 g  0.08-0.8 g                                      S       0.1 g     0.1-8 g     0.2-5 g                                         Fe      6 mg      6-140 mg    9-80 mg                                         Zn      2 mg      2-100 mg    3-40 mg                                         Cu      0.6 mg    0.6-16 mg   1-10 mg                                         Mn      0.6 mg    0.6-20 mg   0.9-8 mg                                        ______________________________________                                    

Sulfur desirably is employed in the form of sulfate. Some of the metalsrequired are advantageously added in the form of a sulfate, so that theminimum concentrations of sulfur normally are exceeded. Any or all ofthe metals listed can be used or present as the sulfate. Preferably, themagnesium, calcium, iron, zinc, copper, and manganese are employed inthe form of a sulfate or chloride, or in the form of a compound which isconverted in situ to a sulfate or chloride. The potassium preferably isemployed as a sulfate, chloride, or phosphate or in the form of acompound which is converted in situ to a sulfate, chloride, orphosphate. The phorphorus preferably is employed in the form ofphosphoric acid or in the form of a phosphate, monohydrogen phosphate,or dihydrogen phosphate, e.g., as a potassium or ammonium salt, or as acompound which is converted in situ to such a salt.

Conveniently, a primary mineral salts medium can be employed to includethe nutrients comprising P, K, Mg, S, and Ca; and a trace mineral mediumto supply nutrients comprising Fe, Zn, Mn, and Cu.

Other elements which may be present, at least in trace amounts, includesuch as sodium and cobalt, e.g., as a halide or sulfate; molybdenum,e.g., as molybdate; boron, e.g., as borate; selenium, e.g., as seleniteor selenate; or iodine, e.g., as iodide.

In typical high cell density fermentation, the ferment will compriseabout one-half supernatant medium and one-half yeast cells, by volume.These one-half by volume yeast cells, however, will contain at leastabout two-thirds of the mineral salts content of the ferment (liquidplus cells).

YEAST

According to my process, I employ a culture of a yeast suitable forgrowth on carbon-containing substrates under aqueous fermentationconditions. Suitable yeasts include species from the genera Candida,Hansenula, Torulopsis, Saccharomyces, Pichia, Debaryomyces, andBrettanomyces. The presently preferred genera include Candida,Hansenula, Torulopsis, Pichia, and Saccharomyces. Examples of suitablespecies include:______________________________________Brettanomycespetrophilium Pichia farinosaCandida boidinii Pichia polymorphaCandidalipolytica Pichia membranaefaciensCandida mycoderma Pichia pinusCandidautilis Pichia pastorisCandida stellatoides Pichia trehalophilaCandidarobusta Saccharomyces cerevisiaeCandida claussenii SaccharomycesfragilisCandida rugosa Saccharomyces roseiCandida tropicalisSaccharomyces acidifaciensDebaryomyces hansenii SaccharomyceselegansHansenula minuta Saccharomyces rouxiiHansenula saturnusSaccharomyces lactisHansenula californica Torulopsis sonorensisHansenulamrakii Torulopsis candidaHansenula silvicola Torulopsis bolmiiHansenulapolymorpha Torulopsis versatilisHansenula wickerhamii TorulopsisglabrataHansenula capsulata Torulopsis molishianaHansenula glucozymaTorulopsis nemodendraHansenula henricii Torulopsisnitratophila,Hansenula nonfermentans Torulopsis pinus, andHansenulaphilodenra Torulopsis bombicolaHansenulaholstii.______________________________________

If desired, mixtures of two or more species of yeasts can be employed.The particular yeast employed depends in part on the carbon-containingsubstrate to be used since it is well known that different yeasts oftenrequire somewhat different substrates for best growth; or, that specificbiochemical conversions are best conducted by specific yeast genera orspecies. For example, it is recognized that some particular strains ofspecies listed above, such as of Pichia pastoris, do not grow onmethanol, i.e., do not utilize methanol.

It should be kept in mind that the utilization of yeasts for variouschemical conversions sometimes is considered in a simplistic fashion asmerely full or partial oxidation. In fact, however, yeast conversion ofthe various possible substrates involves complex and complicatedenzymatic conversion pathways through a variety of intermediates. Hence,the beauty and variety of aqueous biochemical/enzymatic conversions isvery large. My high salts/high cell density method improves allyeast-based conversions.

Presently preferred for single cell protein production are those Pichiapastoris and Hansenula polymorpha which do grow suitably onoxygenated-hydrocarbon feedstocks, particularly, a lower alcohol such asmethanol. Presently preferred are the particular strains designated as,or which are derived from, the strains deposited as Pichia pastoris(Culture 21-2) NRRL Y-11431, Pichia pastoris (Culture 21-1) NRRLY-11430, Hansenula polymorpha NRRL Y-11170, and Hansenula polymorpha(Culture 21-3) NRRL Y-11432, since I have found these strains to beparticularly suitable for use in producing SCP protein materials at highcell densities with high yields. This feature of these strains of Pichiapastoris (Culture 21-2) NRRL Y-11431 and Pichia pastoris (Culture 21-1)NRRL Y-11430 is considered particularly unusual for these species sinceout of four Pichia pastoris cultures tested, only two would grow onmethanol at all. I consider Pichia pastoris (Culture 21-2) NRRL Y-11431,Pichia pastoris (Culture 21-1) NRRL Y-11430, and Hansenula polymorpha(Culture 21-3) NRRL Y-11432, also to be novel and unique. Pichiapastoris NRRL Y-11430 and Y-11431 further are particularly preferred foruse when the coproduction of a particularly useful alcohol oxidase alsois desired.

The carbon energy substrate can be any carbon energy source, such ashydrocarbons, oxygenated hydrocarbons, including various carbohydrates,and the like, suitable as yeast substrates. It is recognized thatparticular yeasts do vary in their preference for various substrates.

The presently preferred substrates for aqueous fermentation conditionsfor yeasts are the carbon-oxygen-hydrogen significantly water-solublecompounds. The term "oxygenated hydrocarbon" is intended to be a genericterm in this disclosure descriptive of compounds employable, and notnecessarily a limiting term referring to the source of the substrate.For this disclosure, the oxygenated hydrocarbons include thewater-soluble carbohydrates, as well as those alcohols, ketones, estes,acids, and aldehydes, and mixtures, which are reasonably significantlywater-soluble in character generally of 1 to 20 carbon atoms permolecule. The more suitable oxygenated hydrocarbons are those ofsubstantially greater water-solubility of up to about 10 carbon atomsper molecule, or are the water-soluble carbohydrates generally.

Exemplary carbohydrates include glucose, fructose, galactose, lactose,sucrose, starch, dextrin, and the like, alone or in admixture. Of theother types of oxygenated hydrocarbons, examples include methanol,ethanol, ethylene glycol, propylene glycol, 1-propanol, 2-propanol,glycerol, 1-butanol, 2-butanol, 3-methyl-1-butanol, 1-pentanol,2-hexanol, 1,7-heptanediol, 1-octanol, 2-decanol, 1-hexadecanol,1-eicosanol, acetone, 2-butanone, 4-methyl-2-pentanone, 2-decanone,3-pentadecanone, 2-eicosanone, formaldehyde, acetaldehyde,propionaldehyde, butyraldehyde, hexanal, 7-methyloctanal, tetradecanal,eicosanal, acetic acid, propionic acid, butyric acid, glutaric acid,5-methylhexanoic acid, azelaic acid, dodecanoic acid, eicosanoic acid,methyl formate, methyl acetate, ethyl acetate, propyl butyrate,isopropyl hexanoate, hexyl 5-methyloctanoate, octyl dodecanoate, and thelike, as well as mixtures thereof.

It also is possible to employ in accordance with my process, thoughpresently less preferred where single cells are the desired product foruse for such as food because of the difficulty sometimes encountered inremoving residual substrate from the single cell protein cells, normalparaffins of such as 10 to 20 carbon atoms per molecule. Yeastsgenerally do not assimilate paraffins of less than 10 carbon atoms permolecule. These typically include such as decane, undecane, dodecane,tridecane, tetradecane, pentadecane, hexadecane, octadecane, eicosane,and the like, and mixtures thereof.

Presently preferred for yeast cell production are the water-solublealcohols of 1 to 4 carbon atoms, water-soluble acids of 2 to 4 carbonatoms, and the water-soluble carbohydrates. Preferred are thewater-soluble monohydric aliphatic hydrocarbyl alcohols. It should benoted that 2-methyl-1-propanol is inhibitory to some yeasts, and infermentations with such yeasts this alcohol should be avoided. Presentlymost preferred are the alcohols of 1 to 4 carbon atoms (other than2-methyl-1-propanol); of these methanol and ethanol presently arepreferred over the others; and methanol is the most prefered, due to thelow relative cost of such feedstock.

Petroleum gases can be oxidized, and the water-soluble materialsemployed, such as oxidation of methane, ethane, and the like, to providemixtures predominantly of the corresponding alcohol as well as variousaldehydes, ketones, acids, and the like, and similarly suitablehydrocarbon fractions from various petroleum refinery sources producedwithin the integrated refining and chemical processing complex,sometimes termed a petrocomplex, can be utilized for fermentationpurposes.

The salts in the supernatant are at a relatively low concentration,since there is a high take-up by the growing reproducing cells. Themineral salts in the cells may not be as fed or applied since some maybe in a bound organic form. Mineral analysis of the ferment, of course,would reflect a total mineral content.

In addition to the mineral salts, vitamins (organic growth factors) canbe employed in the ferment as is known in the art, when their presenceis desirable for the propagation of the particular yeast chosen. Forexample, many yeasts for their proper propagation, seem to require thepresence of one or both of the vitamins biotin and thiamine, or othermedium constituents which contain these vitamins, e.g., yeast extract.Thus, for example, with a yeast such as a Hansenula polymorpha, it isdesirable to employ biotin in an amount of about 0.04 to 0.8 milligramper liter of aqueous mineral medium and thiamine hydrochloride in anamount of about 4 to 80 milligrams per liter of aqueous mineral medium.Alternatively, all or part of the biotin and thiamine can be provided byuse of yeast extract or the like.

In my application Ser. No. 875,667 filed Feb. 6, 1978, now U.S. Pat. No.4,226,939 issued Oct. 7, 1980, I disclose that the employment of watercontaining residual amounts of chlorine, such as is commonly encounteredin water from purification treating processes in some countries, inpreparing mineral medium to which growth factors are added for use inaqueous aerobic fermentation processes tends to render ineffective thegrowth factors, particularly vitamins such as biotin or thiamine. I alsodisclose my discovery that in such fermentation system, employing anaqueous mineral medium prepared from water containing residual chlorine,that removal of the chlorine before adding the growth factors avoids theloss or deactivation of the growth factors. Thus, I also diclose methodsof treating the residual chlorine-containing water so as to effectivelyeliminate the traces of residual chlorine therefrom and thus avoid thevitamin loss.

I have now found that water containing heretofore objectionable amountsof residual chlorine nevertheless can be utilized in fermentationprocesses employing vitamins yet without inactivation of the vitamins bythe chlorine if the vitamins are added to the fermentation zone as aseparate stream separate from the aqueous nutrient medium stream. Thus,the mineral nutrient medium can now employ make-up water even containingtrace amounts of chlorine. This arrangement thus avoids the need forpre-treating, by expensive and/or time consuming methods, the waterwhich contains residual trace amounts of chlorine.

The above-described separate addition of the vitamins to thefermentation zone is preferably and conveniently accomplished byadmixing the vitamins with at least a portion of but preferably theentire carbon energy substrate stream prior to charging these materialsto the fermentation zone. If an aqueous admixture of vitamins and carbonenergy substrate is employed, the water used for initial dilution of thevitamins should preferably be free of traces of residual chlorine, suchas deionized water, to avoid any premature loss before mixing with theaqueous carbon substrate stream such as methanol-in-water.

If desired, and also preferred, an admixture can be made of water and awater-soluble carbon substrate such as methanol, such as about 20 volumepercent methanol in water, and then the vitamins can be dissolved in themethanol-in-water solution, and fed then to the fermentor. By this mode,residual chlorine need not be first removed, but yet the vitamins arefully preserved.

In a more preferred embodiment, the separate addition of vitamins to thefermentation zone is accomplished utilizing an admixture of vitamins, atleast a portion of the carbon energy substrate as noted above and thefurther addition of an aqueous trace mineral salts solution. The tracemineral salts comprise what has been referred to hereinabove as thetrace elements such as cobalt, molybdenum, boron, selenium, iodine, aswell as manganese, copper, zinc, and iron. The use of this morepreferred embodiment not only avoids the vitamin inactivation problemcaused by traces of chlorine in the water used for the aqueous mineralsalts medium, but also avoids another problem that is often encounteredin the fermentation processes. This problem is the formation ofprecipitates in the heat sterilization zone employed to treat theaqueous mineral salts medium, requiring frequent cleaning. The presenceof the trace mineral salts in its usual admixture with the primarymineral nutrient salts apparently promotes the formation of troublesomeprecipitates in the heat sterilization zone. Thus, by not including thetrace mineral salts in the aqueous mineral salts medium stream, butrather instead charging the trace mineral salts in admixture with thevitamins and at least a portion of the carbon energy substrate solvestwo very troublesome problems. As noted above, the water used to preparethe admixture of trace mineral salts, at least a portion of the carbonenergy substrate, and the vitamins should preferably be free of residualtraces of chlorine.

The stream comprised of vitamins, a portion of the carbon energysubstrate, and trace minerals can be sterilized by filtration ifdesired. However, it is preferable and convenient to combine said streamwith the major carbon energy substrate stream prior to charging to thefermentation zone and filtering the entire combined streams just priorto chargint to the fermentation zone.

The fermentation itself is an aerobic aqueous process requiringmolecular oxygen which is supplied by a molecular oxygen-containing gassuch as air, oxygen-enriched air, or even substantially pure molecularoxygen, so as to maintain the ferment with an oxygen partial pressureeffective to assist the microorganism species in growing or inbiochemically converting substrate in a thriving fashion. By using anoxygenated hydrocarbon substrate, the total oxygen requirements forgrowth or substrate conversion of the microorganism are reduced from therequirements when a paraffin is used. Even so, adequate quantities ofmolecular oxygen must be supplied for growth, since the assimilationand/or bioconversion of the substrate and corresponding growth of themicroorganism is, in part, a combustion process.

The rate at which molecular oxygen is supplied to the forment should besuch that the growth of the yeast or substrate conversion is not limitedby lack of oxygen. Fermentor designs vary widely in their ability totransfer oxygen to the culture. Although the overall aeration rates canvery over a considerable range, with fermentors that are very efficientin oxygen transfer aeration generally is conducted at a rate of about0.5 to 8, preferably about 1 to 6, volumes (at the pressure employed andat 25° C.) of molecular oxygen-containing gas per liquid volume in thefermentor per minute. This amount is based on air of normal oxygencontent being supplied to the reactor, and in terms of pure molecularoxygen the respective ranges would be about 0.1 to 1.7, or preferablyabout 0.2 to 1.3, volumes (at the pressure employed and at 25° C.) ofmolecular oxygen per liquid volume in the fermentor per minute.

The pressure employed for the microbial fermentation step can rangewidely. Typical pressures are about 0 to 150 psig, presently preferablyabout 0 to 60 psig, more preferably 35 to 40 psig, as a balance ofequipment and operating costs versus oxygen solubility achieved. Greaterthan atmospheric pressures are advantageous in that such pressures dotend to increase the dissolved oxygen concentration in the aqueousferment, which in turn can help increase cellular growth rates. At thesame time this is counterbalanced by the fact that high pressures doincrease equipment and operating costs.

The fermentation temperature can vary somewhat, but generally will beabout 25° C. to 65° C., generally preferalby about 28° C. to 50° C. Theyeast cultures Pichia pastoris Culture 21-2 deposited as NRRL Y-11431and Pichia pastoris Culture 21-1 deposited as NRRL Y-11430 generallyprefer a ferment temperature of about 30° C. The Hansenula polymorphaNRRL Y-11170 and Hansenula polymorpha Culture 21-3 deposited as NRRLY-11432 presently appear to prefer a ferment temperature on the order ofabout 38° C. to 40° C.

Yeasts require a source of assimilable nitrogen. The assimilablenitrogen can be supplied by any nitrogen-containing compound orcompounds capable of releasing nitrogen in a form suitable for metabolicutilization by the yeast microorganism. While a variety of organicnitrogen source compounds, such as protein hydrolysates, technically canbe employed, usually cheaper nitrogen-containing compounds such asammonia, ammonium hydroxide, urea, and various ammonium salts such asammonium phosphate, ammonium sulfate, ammonium pyrophosphate, andammonium chloride can be utilized. Ammonia gas itself is convenient forlarge scale operations, and can be employed by bubbling through theaqueous microbial ferment in suitable amounts. At the same time, suchammonia also assists in pH control.

The pH range in the aqueous microbial ferment should be in the range ofabout 3 to 7, more preferably and usually about 3.5 to 5.5. Preferencesof certain microorganisms for a pH range are dependent to some extent onthe medium employed, as well as on the particular microorganism, andthus may change somewhat with change in medium as can be readilydetermined by those skilled in the art.

The average retention time of the ferment in the fermentor can varyconsiderably, depending in part on the fermentation temperature andyeast culture employed. Generally, the retention time will be about 2 30hours, preferably presently about 4 to 14 hours, based on averageretention.

High concentrations of some of the described carbon and energysubstrates, particularly such as methanol or formaldehyde or the like,may be inhibitory to satisfactory microbial growth or even toxic to themicroorganisms in the fermentation. Relatively high concentrations ofsuch substrates thus should be avoided, so that it is generallydesirable to maintain the substrate concentration in the ferment at amaximum tolerable level. With some of the lower alcohols, this level inthe ferment generally is about 0.001 to 5 volume percent, preferablyabout 0.005 to 0.05 volume percent, while with the aldehydes the levelshould be one-tenth of these due to the toxicity of aldehydes, so as toneither starve nor inhibit the growth rates of the microorganismschosen.

When the carbon and energy source material contains an aldehyde inamounts potentially deleterious to the microorganism, the deleteriousaldehyde effects can be allievated by first treating the substrate witha suitable amount of a nitrogen-containing compound, preferably ammonia,ammonium hydroxide, or other active ammonium compound, in a ratio ofabout 0.01 to 10 mol equivalents of such nitrogen-containing compoundsper mol of aldehyde. Such a treated substrate then is not only thecarbon energy source, but also contains at least a portion of thenecessary assimilable nitrogen.

Conveniently, the fermentation is conducted in such a manner that thecarbon-containing substrate can be controlled as a limiting factor,thereby providing good conversion of the carbon-containing substrate toyeast cells and extracellular products, thereby avoiding potentialcontamination of the yeast cells with a substantial amount ofunconverted substrate. The latter is not a problem with water-solublesubstrates, since any remaining traces are readily washed off. It may bea problem, however, in the case of non-water-soluble substrates such asthe higher n-paraffins, requiring added product treatment steps such asremoval of residual hydrocarbon by suitable solvent washing steps.

Continuous operation is much to be preferred for ease of control,production of uniform quantities of either cells or extracellularproducts, and most economical uses of all equipment. In a continuousprocess, the carbon and energy source material as substrate, aqueousmineral medium, assimilable nitrogen source, and molecularoxygen-containing gases, are added continuously to the ferment in thefermentor combined with continuous withdrawal of ferment. Although thevolume ratio of added carbon energy substrate:added aqueous mineralmedium can vary over a wide range, depending in part on the nature ofthe carbon-containing substrate, generally it will be in the range ofabout 1:9 to 6:4, presently and preferably in the range of about 2:8 to5:5.

One skilled in the art readily recognizes that the maximum cell densityobtainable is a function of the cell yield (g of cells per g ofsubstrate feed) and the percent substrate in the total feed to thefermentor. This, Pichia pastoris NRRL Y-11430, which gives approximately40 percent yield of single cells when grown on methanol as carbonsource, exhibits a maximum cell density of about 65 grams of cells perliter of ferment from a total feed including 20 volume percent methanol.Yet, with a total feed including 40 volume percent methanol, the samePichia pastoris exhibits a maximum cell density of greater than 126grams of cells per liter of ferment. Total feed is the carbon substrateplus mineral media including water.

If desired, part or all of the carbon energy source material and/or partof the assimilable nitrogen source such as ammonia can be added to theaqueous mineral medium prior to passing the aqueous mineral medium tothe fermentor. Most convenient in my work in high cell densityfermentations employing methanol to produce single cell protein has beenthe use of a feed ratio of about 40 volume percent alcohol to 60 volumepercent mineral salts medium.

Each of the streams introduced into the reactor preferably is controlledat a predetermined rate, or in response to a need determinable bymonitoring, such as concentration in the ferment of the carbon energysubstrate, the pH, the dissolved oxygen, the cell density measurable bylight transmittancy, or the like, and the oxygen or carbon dioxide inthe off-gases from the fermentor. The feed rates of the variousmaterials streams can be varied so as to obtain as rapid a cell growthrate as possible, constant with efficient utilization of the carbon andenergy source, to obtain as high a yield of yeast cells and/orextracellular products, relative to substrate charge as possible, or tomaximize the particular biochemical conversion being practiced. Thus, bythe process of my invention, yeast cells can be obtained in yields ofabout 30 to 110 grams per 100 grams substrate charged, depending in parton the particular substrate used.

All equipment, reactor, or fermentation means, vessel or container,piping, attendant circulating or cooling devices, and the like, mostpreferably are sterilized, usually by employing steam such as at about250° F. (121° C.) for at least about 15 minutes. The sterilized reactoris inoculated with a culture of the specified microorganism in thepresence of all the required nutrients, including molecular oxygen, andthe carbon-containing substrate.

The type of fermentor employed is not critical in the practice of thefermentation process of my invention, though presently preferred isoperation in a foam-filled fermentor mode. A fermentor designed toencourage and maintain the produced foam usually is beneficial to theprocess of achieving the increased oxygen transfer necessary to maintaindesired high cell densities and rapid growth rates.

The typical start-up procedure from stock culture to continuous culturemay take a number of days and involve several steps. For example, theinitial growth of a stock yeast culture on YM media conveniently employsa readily assimilable carbon source such as glucose, and takes about 2days. Cells are then transferred to small flasks containing minimalnutrient medium with the desired carbon source, such as methanol orother. Usually, 2-3 days are required for this phase. Cells are thentransferred into larger flasks with fresh nutrient medium and additionalcarbon source. About 3 more days usually are necessary for the cultureto grow up in this medium. There is then enough culture to inoculate atypical 14 L laboratory fermentor.

In starting out a continuous fermentation, the aqueous mineral medium,suitable concentration of carbon source, assimilable nitrogen, tracecomponents where desired, and the starting inoculum of the desired yeaststrain, the latter selected in accordance with the type of biochemicalconversion to be practiced, and grown up as described above, are placedin a sterilized fermentor, and suitable flows of oxygen and the variousfeeds are gradually commenced. If desired, the initial fermentationsubstrate can be such as glucose or glycerol, with gradual change tosuch as methanol as cell density builds up. It is possible to begin atlow mineral salts levels in the aqueous ferment and build up to a highmineral salts level by feeding an aqueous mineral medium having a highconcentration of mineral salts to the ferment, through I normally simplyadd high salts medium initially to the fermentor to commence immediateoperation. One skilled in the art realizes that a brief lag time willusually occur at start-up before the inoculum builds up enough cells forfull input of salts and substrate to be effectively utilized.

PRODUCT RECOVERY

The yeast cells produced in accordance with my high salts/high celldensity process can be recovered from the fermentation admixtureeffluent by conventional means, such as by centrifugation or filtration.Extracellular products can be recovered from the substantially cell-freeremaining supernatant liquid by conventional means. The substantiallycell-free effluent can be treated, for example, with acetone or a loweralcohol such as methanol or ethanol to precipitate polymeric materialsproduced extracellularly. The cell-free effluent also can be treated bysolvent extraction and/or base extraction to recover, if desired, otherextracellular products such as pigments, vitamins, or organic acidsproduced during the culturing process. The cell-free effluent, with orwithout such intervening treatment, can be returned to the fermentor asa part of the aqueous makeup, or as a substantial or almost total partof the aqueous makeup, to avoid waste disposal problems insofar aspossible.

The microbial cells usually are killed if desired, by heat or chemicalmeans, and this can be done before or after the separation of the cellsfrom the fermentor effluent. The yeast cells are a valuable source ofprotein for man as well as beast. For human consumption, the cells canbe treated as necessary to reduce the nucleic acid, but for animal feedpurposes such tretment does not appear presently necessary.

The yeast cells are, in addition, a valuable source of enzymes forcarrying out chemical conversions. In such cases, a broth of cells grownto high cell density according to the process of my invention can thenbe maintained in a resting state with no further manipulation requiredsuch as concentration step or the like in order to utilize the cells forcarrying out enzyme conversions. Alternately, the dense broth of cellsobtained in the process of my invention can be directly subjected toimmobilization techniques such as described in "Methods in Enzymology",Vol. XLIV K. Moshach, ed., pp. 11-332. By achieving a high cellulardensity in the fermentation step, one avoids the need to concentrate thecells before immobilization, and thus the possibility of proteindenaturation or loss of extracellular enzymes is greatly reduced. Forexample, the conversion of C₃ -C₆ secondary alcohols to ketonesdisclosed by Hou, Patel and Laskin in U.S. Pat. Nos. 4,250,259,4,268,630, and 4,269,940, is enhanced in the presence of the largenumber of cells obtained in the practice of my high cell densityfermentation process, compared to the number of active cells made in atypical fermentation process.

Production of a high cell density broth is desirable whenever the yeastcells are to be used to promote secondary conversions, such as theoxidative reactions referred to above. Other examples are found inChemical Oxidations With Microorganisms by G. S. Fonken and R. A.Johnson, (Dekker, N.Y., 1972). Anaerobic production of ethanol fromglucose is most efficiently carried out by first producing a largenumber of cells in an aerobic fermentation, followed by the anaerobicconversion of glucose to ethanol (D. Williams and D. M. Munnecke,Biotech. and Bioeng. 23, 1813-1825 (1981). This overall conversion isenhanced by first producing the maximum feasible number of cellspossible per liter of ferment in the first or aerobic stage, such as byemploying my inventive high cell density method.

The oxidation of n-paraffins to diacids as described by Wegner in U.S.Pat. No. 3,796,630 is another application in which my high cell densitymethod can be beneficial. Cells are grown on glucose or sucrose ascarbon source and n-paraffins are oxidized at the same time to diacids.The more yeast cells produced, the greater the conversion of n-paraffinsto diacids.

In accordance with my invention, employing high cell density operation,e.g., a cell density within the range of about 60 to 160, preferablyabout 70 to 150, grams of yeast cells, on a dried basis, per liter offerment, can be obtained in high yield. If desired, the cells can berecovered from the fermentation admixture by centrifugation or otherseparation means. Also, if desired, the concentrated cells then can bewashed such as by mixing with water, and separated such as byrecentrifuging, or by adding water prior to or during centrifugation tosubstantially free the cells of mineral medium, and the washingsincluding the separated mineral medium then can be returned to thefermentor as water and mineral medium makeup, thus substantiallyreducing or avoiding waste disposal problems. The recovered cells thencan be employed in a chemical conversion process as described above, orsimply dried to produce a dried product for future use. If desired, thehigh cell density fermentor effluent in total can be dried to produce awhole dried product of dried cells and residual water soluble substancesincluding salts, and this whole-dried product used as a very usefulanimal feed of high protein-high salts character.

EXAMPLES

The following are descriptive runs employing the process in accordancewith my discovery. Particular amounts of materials, or particular typesof feedstocks employed, particular species or strains of yeast, shouldbe considered as illustrative and not as limitative of my invention.

EXAMPLE I

In a run conducted under continuous aerobic fermentation processconditions, methanol and an aqueous mineral salts medium in a volumeratio of 30.15 to 69.85, respectively, were fed individually to afermentor inoculated with the yeast Pichia pastoris Culture 21-2deposited as NRRL Y-11431. No pre-conditioning medium or substrate wasemployed. The fermentor was a 4-liter fementor with a 2-liter liquidvolume, with automatic pH, temperature, and level control. Agitation wasprovided by two impellers rotating at 1000-1200 rpm. The aeration ratewas 1-1.5 volumes (at about atmospheric pressure and about 25° C.) pervolume of ferment per minute of air supplemented with an includingsufficient oxygen to maintain in the fermentation mixture an amount ofdissolved oxygen equal to about 20 percent of that which would bedissolved in the fermentation mixture saturated with air at atmosphericpressure and about 30° C. Aqueous ammonium hydroxide (from 2 partsconcentrated ammonium hydroxide and 1 part deionized water, by volume)was added at such a rate as to maintain the pH of the fermentationmixture at about 3.5.

The aqueous mineral salts medium employed was prepared by mixing, foreach liter of solution, 12.5 mL 85 percent H₃ PO₄, 2.5 g 85 percent KOH,8.5 g KCl, 7.0 g MgSo₄.7H₂ O, 1.5 g CaCl₂.2H₂ O, 25 mL of trace mineralsolution A, 25 mL of trace mineral solution B, 10 mL of abiotin-thiamine hydrochloride solution, about 0.08 mL of antifoam agent(Mazu DF-37C), and sufficient deionized water to make 1 liter ofsolution.

Trace mineral solution A was prepared by mixing, for each liter ofsolution, 4.8 g FeCl₃.6H₂ O, 2.0 g ZnSO₄.7H₂ O, 0.02 g H₃ BO₃, 0.20 gNa₂ MoO₄.2H₂ O, 0.30 g MnSO₄.H₂ O, 0.08 g KI, 0.06 g CuSO₄.5H₂ O, 3 mLconc. H₂ SO₄, and sufficient deionized water to make 1 liter ofsolution.

Trace mineral solution B was prepared by mixing, for each liter ofsolution, 2.0 g FeCl₃.6H₂ O, 2.0 g ZnSO₄.7H₂ O, 0.3 g MnSO₄.H₂ O, 0.6 gCuSO₄.5H₂ O, 2 mL conc. H₂ SO₄, and sufficient deionized water to make 1liter of solution.

The biotin-thiamine hydrochloride solution was prepared by mixing 2 mgbiotin, 200 mg thiamine hydrochloride, and 50 mL deionized water.

The fermentation was conducted at about 30° C. and about atmosphericpressure, with a retention time of 7.0 hours.

Yeast cells were separated from the fermentation effluent bycentrifugation, washed by suspension in water and recentrifugation,dried overnight at 100° C., and weighed. On a dried basis, the yeastcells were produced in a yield of 42.3 g per 100 g of methanol fed, thecell density being at the high level of 100.7 g of cells per liter ofeffluent.

EXAMPLE II

A further fermentation run was conducted using essentially the proceduredescribed in Example I except that the composition of the aqueousmineral salts medium was somewhat different, the volume ratio ofmethanol to the aqueous mineral salts medium was 40.8 to 59.2, theaqueous ammonium hydroxide for pH control was prepared from 3 partsconcentrated ammonium hydroxide and 1 part deionized water, by volume,and the fermentation retention time was 8.35 hours.

The aqueous mineral salts medium for use in this run was prepared bymixing, for each liter of solution, 20.0 mL 85 percent H₃ PO₄, 4.0 g 85percent KOH, 12.0 g KCL, 10.4 g MgSO₄.7H₂ O, 2.4 g CaCl₂.2H₂ O, 40 mL ofthe trace mineral solution A as described in Example I, 40 mL of thetrace mineral solution B as described in Example I, 16 mL of thebiotin-thiamine hydrochloride solution as described in Example I, about0.08 mL of antifoam agent, and sufficient deionized water to make 1liter of solution.

Yeast cells were separated from the fermentation effluent, washed, anddried as in Example I. On a dried basis, the yeast cells were producedin a yield of 41.4 g per 100 g of methanol fed, the cell density beingat the very desirably high level of 133.3 g of cells per liter ofeffluent.

EXAMPLE III

A continuous aerobic fermentation process was conducted in the fermentoras described in Example I, this time inoculated with the yeast speciesHansenula polymorpha Culture 21-3 deposited as NRRL Y-11432. To thefermentor was fed a mixture of methanol and aqueous mineral salts mediumcontaining 300 mL methanol per liter total solution. The stirredfermentation mixture was aerated by passing into the fermentor 2 volumes(at about atmospheric pressure and about 25° C.) per volume of fermentper minute of air supplemented with and including sufficient oxygen tomaintain in the fermentation mixture an amount of dissolved oxygen equalto about 20 percent of that which would be dissolved in the fermentationmixture saturated with air at atmospheric pressure and about 38° C.

Aqueous ammonium hydroxide (from 2 parts concentrated ammonium hydroxideand 1 part deionized water, by volume) was added at a rate to maintainthe pH of the fermentation mixture at 3.7 to 4.1.

The mixture of methanol and aqueous mineral salts medium was prepared bymixing, for each liter of solution, 300 mL methanol, 6 mL 85 percent H₃PO₄, 3 g KCl, 4.5 g MgSO₄.7H₂ O, 0.6 g CaCl₂.2H₂ O, 0.3 g NaCl, 10 mL oftrace mineral solution A as described in Example I, 10 mL of tracemineral solution B as described in Example I, 4 mL of thebiotin-thiamine hydrochloride solution described in Example I, 4 dropsof antifoam agent, and sufficient deionized water to make 1 liter ofsolution.

The fermentation was conducted at about 38° C. and about atmosphericpressure, with a retention time of 5.66 hours.

Yeast cells were separated from the fermentation effluent, washed, anddried as in Example I. On a dried basis, the yeast cells were producedin a yield of 31.0 g per 100 g of methanol fed, the cell density beingat 73.3 g of cells per liter of effluent.

EXAMPLE IV

In a continuous aerobic fermentation process, methanol and an aqueousmineral medium in a volume ratio of 36.9 to 63.1, respectively, were fedindividually to a fermentor inoculated with the yeast species Pichiapastoris Culture 21-1 deposited as NRRL Y-11430. The fermentor was a1500-liter foam-filled fermentor with a liquid volume of about 600liters, with automatic pH, temperature, and level control, and equippedwith a draft tube. Agitation was provided by a turbine, below the drafttube, driven at 750 rpm. The aeration rate was about 1.6 volumes of air(at about 38 psig and about 25° C.) per volume of ferment in thefermentor per minute. Anhydrous ammonia was added at a rate to maintainthe pH of the ferment mixture at about 3.5.

The primary aqueous mineral salts medium was prepared by mixing, foreach liter of solution, 12.2 mL 75 percent H₃ PO₄, 6.0 g KCl, 6.0 gMgSO₄.7H₂ O, 0.8 g CaCl₂.2H₂ O, 2.0 g 85 percent KOH, 2.0 mL of tracemineral solution C, 0.8 mL of a biotin-thiamine hydrochloride solution,and sufficient tap water to make 1 liter of solution, the tap waterfirst having been treated with enough sodium thiosulfate to react withthe free chlorine present therein.

Trace mineral solution C was prepared by mixing, for each liter ofsolution, 67.5 g FeCl₃.6H₂ O, 18 g ZnSO₄.7H₂ O, 5.0 g MnSO₄.H₂ O, 6.0 gCuSO₄.5H₂ O, 2.0 mL conc. H₂ SO₄, and sufficient deionized water to make1 liter of solution.

The biotin-thiamine hydrochloride solution was made by mixing thecomponents in the ratio of 0.4 g biotin to 40 g thiamine hydrochlorideto 1 liter deionized water.

The fermentation was conducted at 30° C. and 38 psig pressure, with aretention time of 12.7 hours.

Yeast cells were separated from the fermentation effluent bycentrifugation, washed by suspension in water and recentrifugation,dried overnight at 100° C. at 100° C., and weighed. On a dried basis,the yeast cells were produced in a yield of 37 g per 100 g of methanolfed, the cell density being a very desirable 110.3 g of cells per literof effluent.

EXAMPLE V

In a continuous aerobic fermentation process, methanol and an aqueousmineral salts medium in a volume ratio of 40 to 60, respectively, werefed individually to a fermentor inoculated with the yeast species Pichiapastoris Culture 21-1 deposited as NRRL Y-11430. The fermentor was a1500-liter foam-filled fermentor with a liquid volume of about 610liters, with automatic pH, temperature, and level control. Agitation wasprovided by two conventional paddle-type turbines driven at 1000 rpm.The aeration rate was about 4 volumes of air (at about 38 psig and about25° C.) per volume of ferment in the fermentor per minute. Anhydrousammonia was added at such a rate to maintain the pH of the fermentationmixture at about 3.5.

The aqueous mineral salts medium was prepared by mixing, with each literof tap water, 15.86 mL 75 percent H₃ PO₄, 9.53 g K₂ SO₄, 7.8 g MgSO₄.7H₂O, 0.6 g CaCl₂.2H₂ O, and 2.6 g 85 percent KOH. The trace mineralsolution plug biotin was fed separately via the methanol stream at arate of 10 mL per liter of methanol. The trace mineral solution plusbiotin was prepared by mixing 780 mL trace mineral solution D, 20 mLwater, 200 mL methanol and 0.032 g biotin.

Trace mineral solution D was prepared by mixing, for each liter ofsolution, 65 g FeSO₄.7H₂ O, 20 g ZnSO₄.7H₂ O, 3.0 g MnSO₄.H₂ O, 6.0 gCuSO₄.5H₂ O, 5.0 mL conc. H₂ SO₄, and sufficient deionized water to make1 liter of solution.

The aqueous mineral salts medium was fed at a rate of 31.5 liters perhour and the methanol at a rate of 21 liters per hour.

The fermentation was conducted at 30° C. and about 38 psig pressure,with a retention time of 11.6 hours.

For analytical purposes yeast cells were separated from the fermentationeffluent by centrifugation, washed by suspension in water andrecentrifugation, dried overnight at 100° C., and weighed. On a driedbasis, the yeast cells were produced in a yield of 40.6 g per 100 g ofmethanol fed, the cell density being a very desirable 128.4 g of cellsper liter of effluent. The total solids content of the ferment (effluentfrom the fermentor) was 134.7 g per liter, cells plus dissolved solids.The effluent from the fermentor was fed directly through a pasteurizerto kill the yeast and into a spray dryer without further concentrationof treatment.

EXAMPLE VI

In a continuous aerobic fermentation process, methanol and an aqueousmineral salts medium in a volume ratio of 29 to 71, respectively, werefed individually to a fermentor inoculated with the yeast speciesHansenula polymorpha NRRL Y-11170. The fermentor was a 1500-literfoam-filled fermentor with a liquid volume of about 560 liters, withautomatic pH, temperature, and level control, and equipped with a drafttube. Agitation was provided by a turbine, below the draft tube, drivenat 810 rpm. The aeration rate was about 5 volumes of air (at about 38psig and about 25° C.) per volume of ferment in the fermentor perminute. Anhydrous ammonia was added at such a rate as to maintain the pHof the fermentation mixture at about 3.5.

The aqueous mineral salts medium was prepared by mixing 10.38 mL 75percent H₃ PO₄, 4.29 g KCl, 6.44 g MgSO₄.7H₂ O, 0.86 g CaCl₂.2H₂ O, 0.43g NaCl, 3.0 mL of trace mineral solution C, and 0.64 mL of abiotin-thiamine hydrochloride solution in 1000 mL tap water, the tapwater first having been treated with enough sodium thiosulfate to reactwith the free chlorine present therein.

The fermentation was conducted at 39°-40° C. and 38 psig pressure, witha retention time of 7.6 hours.

For analysis yeast cells were separated from the fermentation effluentby centrifugation, washed by suspension in water and recentrifugation,dried overnight at 100° C., and weighed. On a dried basis, the yeastcells were produced in a yield of 33.3 p per 100 g of methanol fed, thecell density being a desirable 76.2 g of cells per liter of effluent.

EXAMPLE VII

A further fermentation run was conducted using essentially the proceduredescribed in Example I except employing a somewhat different compositionof the aqueous mineral salts medium. The inoculum was Pichia pastorisNRRL Y-11430. Ethanol was employed as the carbon source in a volumeratio of 19 to 81 relative to the aqueous mineral medium. Anhydrousammonia was added at such a rate sufficient to maintain the pH of thefermentation mixture at about 3.5. Fermentation retention time was 8.5hours.

The aqueous mineral salts medium for use in this run was prepared bymixing, for each liter of solution, 15.9 mL 75 percent H₃ PO₄, 0.6 gCaSO₄.2H₂ O, 9.5 g K₂ SO₄, 7.8 g MgSO₄.7H₂ O, 2.6 g 85 percent KOH, 5.2mL trace mineral solution E, and about 0.08 mL of antifoam agent (MazuDF-37C).

Trace mineral solution E was prepared by mixing one liter of solutioncomprising: 65 g FeSO₄.7H₂ O, 6.0 g CuSO₄.5H₂ O, 24 g ZnSO₄.7H₂ O, 3.0 gMnSO₄.H₂ O, and 5 mL concentrated H₂ SO₄, then withdrawing 780 ml of thethus prepared solution, adding 0.32 g biotin, and diluting to 1 literwith 200 mL methanol and 20 mL deionized water.

The fermentation was conducted at about 30° C. and about atmosphericpressure. Yeast cells were separated from the fermentation effluent bycentrifugation, washed by suspension in water and recentrifugation,dried overnight at 100° C. and weighed. On a dried basis, the yeastcells were produced in a yield of 72.7 g per 100 g of ethanol fed, thecell density being a very desirable 106.8 g of cells per liter ofeffluent. The total solids content of the ferment (effluent from thefermentor) was 119.3 g per liter, cells plus dissolved solids.

EXAMPLE VIII

A further fermentation run was conducted using essentially the proceduredescribed in Example VII except that glucose was employed as the carbonsource, in a volume ratio of 26 to 74 relative to the aqueous mineralmedium. Fermentation retention time was 9.8 hours.

The same aqueous mineral salts medium described for Example VII wasemployed for this run, except including trace mineral solution D, andomitting the antifoam agent.

The fermentation was conducted at about 30° C. and about atmosphericpressure. Yeast cells were separated from the fermentation effluent bycentrifugation, dried overnight at 100° C. and weighed. On a driedbasis, the yeast cells were produced in a yield of 46.6 g per gram ofglucose fed, the cell density being a very desirable 120.0 g of cellsper liter of effluent. The total solids content of the ferment (effluentfrom the fermentor) was 129.6 g per liter, cells plus dissolved solids.

EXAMPLE IX

In various runs, I have studied the effect of the volume percent carbonsource as fed relative to the aqueous mineral media in the totalfermentor feed on the maximum cell densities obtainable with, forexample, Pichia pastoris NRRL Y-11430, and a specific substrate, such asmethanol. In these studies, I have developed relationships tabulatedbelow for methanol:

                  TABLE II                                                        ______________________________________                                        Relative Volume                                                               % Methanol Expected            Actual Recovered                               in Total Feed                                                                            Cell Density.sup.(1)                                                                      Yield.sup.(2)                                                                         Weight (total solids)                          ______________________________________                                        10          33.2       .42     --                                             20          64.8       .41     --                                             40         126.4       .40     133                                            50         154.1       .39     165                                            ______________________________________                                         .sup.(1) The expected cell density is a calculated value obtained by          multiplying the number of grams of methanol fed per liter of feed times       the yield.                                                                    .sup.(2) gm Single cell product/gm MeOH feed.                            

Yields of about 40 percent are obtained for growth of Pichia pastorisNRRL Y-11430, although yields of single cells are seen to decreaseslightly at higher cell densities.

In order to accommodate the increased substrate feed, the concentrationof mineral medium feed preferably is adjusted. Several factors are to betaken into account, such as the increased number of cells growing athigher cell densities are achieved and the reduced volume of wateremployed for the mineral feed. Thus, as shown below, the mineral mediumfeed that is employed for growth on 40 volume percent relativeproportion of methanol feed may be about 6 times as concentrated as themineral medium feed employed for growth on 10 volume percent relativeproportion of methanol. On further increase of methanol feed to 50volume percent, the mineral medium compositions preferably is adjustedto about 1.5 times the concentration employed with 40 volume percentrelative proportion methanol feed.

                  TABLE III                                                       ______________________________________                                        Mineral Medium Adjustment as a Function of Methanol Feed                      Volume %               Mineral Concentration                                  Methanol  Mineral Medium                                                                             Increase per 10%                                       in Feed   Concentration                                                                              Increase in MeOH Feed                                  ______________________________________                                        10        1            --                                                     20        2.25         2.25                                                   30        3.86         1.71                                                   40        6.00         1.56                                                   50        9.01         1.50                                                   ______________________________________                                         .sup.(3) Relative to 10% MeOH feed.                                      

Simply put, if one considers as a constant the total volume of totalfeed (substrate such as methanol plus aqueous mineral media), it isapparent that as the substrate amount is increased, that the mineralsfed must also increase.

NOVEL YEASTS

Discovery of yeasts with the capability of rapid growth and highproductivity rates is distinctly advantageous.

I have discovered three very unique cultures of yeasts, namely Pichiapastoris my Culture 21-2, deposited as NRRL Y-11431; Pichia pastoris myCulture 21-1, deposited as NRRL Y-11430; and Hansenula polymorpha myCulture 21-3, deposited as NRRL Y-11432, with highly desirable anduseful properties. These unique cultures grow particularly well athigher mineral salts levels and cell densities.

These three unique cultures of yeasts grow effectively with highproductivity on oxygenated hydrocarbon feedstocks, particularly loweralcohols, most preferably methanol or ethanol. This is particularlynoteworthy with regard to the new Pichia pastoris cultures since somecultures of the species Pichia pastoris simply cannot grow on methanol.These unique species are designated as follows:

    ______________________________________                                                        My Strain Depository                                          Culture Name    Designation                                                                             Designation                                         ______________________________________                                        Pichia pastoris 21-2      NRRL Y-11431                                        Pichia pastoris 21-1      NRRL Y-11430                                        Hansenula polymorpha                                                                          21-3      NRRL Y-11432                                        ______________________________________                                    

The designations NRRL Y-11431, NRRL Y-11430, and NRRL Y-11432 reflectthe fact that I have deposited my novel yeast cultures 21-2, 21-1, and21-3 with the official depository, United States Department ofAgriculture, Agricultural Research Service, Northern Regional ResearchLaboratory, Peoria, Ill. 61604, by depositing therein two agar slantcultures of each, and have received from the depository the individualNRRL strain designations as indicated.

These unique cultures have been deposited in accordance with theprocedures of the Department of Agriculture such that progeny of thesestrains will be available during pendency of this patent application toone determined by the Commissioner of Patents and Trademarks to beentitled thereto according to the Rules of Practice in Patent Cases and35 U.S.C. 122. The deposits have been made in accordance with the Patentand Trademark Office practice such that all restrictions on availabilityto the public of progeny of the unique strains will be irrevocablyremoved upon granting of a patent of which these important strains arethe subject, so that these strains will be available to provide samplesfor utilization in accordance with my invention. Thus, culture samplesfrom these deposits or from my cultures from which the deposits weremade provide strains derived from species of my discovery.

My invention provides in one aspect processes for culturing oxygenatedhydrocarbon-assimilating microbial cells belonging to three new culturesor strains of microorganisms under aqueous aerobic culturing conditions.These strains have been classified:

    ______________________________________                                                                    Hansenula                                         Pichia pastoris                                                                              Pichia pastoris                                                                            polymorpha                                        Culture 21-1   Culture 21-2 Culture 21-3                                      NRRL Y-11430   NRRL Y-11431 NRRL Y-11432                                      ______________________________________                                        Divi- Ascomytina   Ascomytina   Ascomytina                                    sion                                                                          Class Hemiascomycetes                                                                            Hemiascomycetes                                                                            Hemiascomycetes                               Order Endomycetales                                                                              Endomycetales                                                                              Endomycetales                                 Family                                                                              Saccharomy-  Saccharomy-  Saccharomy-                                         cetaceae     cetaceae     cetaceae                                      Genus Pichia       Pichia       Hansenula                                     ______________________________________                                    

The novel and unique microorganisms can be further characterized byproperties as shown in the following tabulation:

    ______________________________________                                                                      Hansenula                                                 Pichia pastoris                                                                        Pichia pastoris                                                                          polymorpha                                                Culture 21-1                                                                           Culture 21-2                                                                             Culture 21-3                                              NRRL     NRRL       NRRL                                                      Y-11430  Y-11431    Y-11432                                         ______________________________________                                        Culture                                                                       Property or                                                                   Test Result.sup.(1)                                                           Gram straining                                                                            +          +          +                                           Spore Forming                                                                             +          +          +                                           Aerobic     +          +          +                                           Approx. size, μ                                                                        3-5        3-5        3-5                                         Optimum temps.,                                                                           30         30         38-40                                       °C.                                                                    Optimum pH  3.5-5.5    3.5-5.5    3.5-5.5                                     Growth factors                                                                            Biotin     Biotin     Biotin and                                                                    thiamine                                    Cell appearance                                                                           Colonies turn                                                                            Form       Form                                        on malt     deep tan with                                                                            hat-shaped hat-shaped                                  extract     formation of                                                                             spores     spores                                      agar        hat-shaped                                                                    spores                                                            Colony appearance                                                                         Cream      Cream      Cream                                       on YM agar  colored;   colored;   colored;                                                no pseudomy-                                                                             no pseudomy-                                                                             no pseudomy-                                            celium     celium     celium                                      Assimilation                                                                  of sugars                                                                     Glucose     +          +          +                                           Galactose   W/L                                                               L-Sorbose   -                                                                 Maltose     -                                                                 Sucrose     -                                                                 Cellobiose  -                                                                 Trehalose   -                                                                 Lactose     -                                                                 Melibiose   -                                                                 Raffinose   -                                                                 Melezitose  -                                                                 Inulin      -                                                                 Soluble Starch                                                                            W/L                                                               Xylose      -                                                                 L-arabinose -                                                                 D-arabinose -                                                                 D-Ribose    -                                                                 L-Rhamnose  -                                                                 Ethyl alcohol                                                                             +          +          +                                           Methyl alcohol                                                                            +          +          +                                           Glycerol    +                                                                 Erythritol  -                                                                 Adonitol    -                                                                 Dulcitol    -                                                                 Mannitol    +                                                                 Sorbitol    +                                                                 Methyl-d-glucoside                                                                        -                                                                 Salicin     -                                                                 Inositol    -                                                                 Fermentation                                                                  of sugars                                                                     Glucose     +          +          +                                           Galactose   -                                                                 Sucrose     -                                                                 Lactose     -                                                                 Maltose     -                                                                 Raffinose   -                                                                 Trehalose   -                                                                 Nitrate                                                                       Assimilation                                                                              -                                                                 Urease Activity                                                                           -                                                                 Arbutin split                                                                             -                                                                 Pseudomycelium or                                                             hyphae formed                                                                 on corn meal                                                                              -                                                                 on yeast agars                                                                            -          -          -                                           Guanine plus                                                                              40%                                                               cytosine content                                                              of DNA                                                                        ______________________________________                                         + = positive                                                                  - = negative                                                                  W/L = weak to latent reaction                                                 .sup.(a) = if value not shown, means not determined.                     

Of course, as with all microorganisms, some of the characteristics maybe subject to some variation depending on the medium and particularconditions.

The media recipes shown in my Examples can be used to culture the novelyeast species of my discovery and invention, though they will grow onother than methanol-containing substrates. These novel yeast culturescan be employed not only with the high salts-media feeds as hereindescribed, but also can be employed under conventional fermentationconditions using a medium such as:

    ______________________________________                                        Component             Amount                                                  ______________________________________                                        Medium IM-1                                                                   KH.sub.2 PO.sub.4     5.0 g                                                   MgSO.sub.4.7H.sub.2 O 0.5 g                                                   CaCl.sub.2.2H.sub.2 O 0.2 g                                                   KCl                   0.5 g                                                   (NH.sub.4).sub.2 SO.sub.4                                                                           3.0 g                                                   Biotin                0.04 mg                                                 Thiamine              4.0 mg                                                  Trace mineral solution.sup.(a)                                                                      2.5 mL                                                  Water                 1,000 mL                                                Sterile methanol.sup.(b) to give                                                                    0.5-1.0 vol %                                            .sup.(a) See recipe below.                                                    .sup.(b) Added just prior to use.                                        

    Trace Mineral Solution                                                        CuSO.sub.4.5H.sub.2 O 0.06 g                                                  KI                    0.08 g                                                  MnSO.sub.4.H.sub.2 O  0.3 g                                                   Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                       0.2 g                                                   H.sub.3 BO.sub.3      0.02 g                                                  ZnSO.sub.4.7H.sub.2 O 2.0 g                                                   FeCl.sub.3.6H.sub.2 O 4.8 g                                                   Distilled water       1,000 mL                                                H.sub.2 SO.sub.4 (conc.)                                                                            3 mL                                                    ______________________________________                                    

The disclosure, including data, illustrates the value and effectivenessof my invention. The examples, the knowledge and background of the fieldof the invention, general principles of microbiology, chemistry, andother applicable sciences, have formed the bases from which the broaddescriptions of my invention, including the ranges of conditions andgeneric groups of operant components, have been developed, and whichhave formed the bases for my claims here appended.

I claim:
 1. In a continuous process of biochemical conversion of acarbon energy substrate to products of fermentation comprising yeastcells and extracellular products which comprises culturing at least oneyeast under aerobic aqueous fermentation conditions, in aqueous fermentcomprising a cellular phase and an aqueous extracellular phase, infermentation means employing effective amounts of a yeast-assimilablecarbon energy substrate, assimilable nitrogen source, water, molecularoxygen, and mineral salts, and withdrawing said aqueous ferment aseffluent for recovery therefrom of said products of fermentation,whereinthe improvement comprises feeding said mineral salts to the aqueousferment in amounts sufficient to maintain in said aqueous ferment thefollowing elements in at least the designated weights per liter ofaqueous ferment: P-1.9 g, K-1 g, Mg-0.15 g, Ca-0.06 g, S-0.1 g, Fe-6 mg,Zn-2 mg, Cu-0.6 mg, and Mn-0.6 mg, thereby producing at least oneproduct of fermentation and maintaining a yeast cell density in saidaqueous ferment of at least about 60 to 160 grams, on a dried basis, perliter of said aqueous ferment; said fermentative means being a fermenterfree from physical means to remove liquid medium from said aqueousferment without removing cells.
 2. The process according to claim 1wherein said yeast is selected from the group of genera consisting ofCandida, Hansenula, Torulopsis, Saccharomyces, Pichia, Debaryomyces, andBrettanomyces.
 3. The process according to claim 2 wherein the yeast isselected from the group of genera consisting of Candida, Hansenula,Torulopsis, Pichia, and Saccharomyces.
 4. The process according to claim2 wherein said yeast is selected from the group of species consistingof:

    ______________________________________                                        Brettanomyces petrophilium,                                                                      Hansenula holstii,                                         Candida boidinii,  Pichia farinosa,                                           Candida lipolytica,                                                                              Pichia polymorpha,                                         Candida mycoderma, Pichia membranaefaciens,                                   Candida utilis,    Pichia pinus,                                              Candida stellatoidea,                                                                            Pichia pastoris,                                           Candida robusta,   Pichia trebalophila,                                       Candida claussenii,                                                                              Saccharomyces cerevisiae,                                  Candida rugosa,    Saccharomyces fragilis,                                    Candida tropicalis,                                                                              Saccharomyces rosei,                                       Debaryomyces hansenii,                                                                           Saccharomyces acidifaciens,                                Hansenula minuta,  Saccharomyces elegans,                                     Hansenula saturnus,                                                                              Saccharomyces rouxii,                                      Hansenula californica,                                                                           Saccharomyces lactis,                                      Hansenula mrakii,  Torulopsis sonorensis,                                     Hansenula silvicola,                                                                             Torulopsis candida,                                        Hansenula polymorpha,                                                                            Torulopsis bolmii,                                         Hansenula wickerhamii,                                                                           Torulopsis versatilis,                                     Hansenula capsulata,                                                                             Torulopsis glabrata,                                       Hansenula glucozyma,                                                                             Torulopsis molishiana,                                     Hansenula henricii,                                                                              Torulopsis nemodendra,                                     Hansenula nonfermentans,                                                                         Torulopsis nitratophila,                                   Hansenula philodendra,                                                                           Torulopsis pinus, and                                                         Torulopsis bombicola.                                      ______________________________________                                    


5. The process according to claim 4 maintaining in each liter of saidaqueous ferment: P 1.9 to 20 g, K 1 to 20 g, Mg 0.15 to 3 g, Ca 0.06 to1.6 g, S 0.1 to 8 g, Fe 6 to 140 mg, Zn 2 to 100 mg, Cu 0.6 to 16 mg,and Mn 0.6 to 20 mg.
 6. The process according to claim 5 maintaining ineach liter of said aqueous ferment about: P 2.2 to 10 g, K 1.5 to 10 g,Mg 0.3 to 1.2 g, Ca 0.08 to 0.8 g, S 0.2 to 5 g, Fe 9 to 80 mg, Zn 3 to40 mg, Cu 1 to 10 mg, and Mn 0.9 to 8 mg.
 7. The process according toclaim 5 wherein said aqueous fermentation conditions includefermentation temperature in the range of 25° C. to 65° C., pH in therange of about 3 to 7, pressure in the range of about 0 to 150 psig, andfermentation time in the range of about 2 to 30 hours based on averageretention.
 8. The process according to claim 7 maintaining a celldensity of about 70 to 150 grams, on a dried basis, per liter of aqueousferment.
 9. The process according to claim 7 maintaining a cellularyield of about 30 to 110 grams per 100 grams substrate charged.
 10. Theprocess according to claim 7 wherein said carbon energy substrate is analcohol of 1 to 4 carbon atoms.
 11. The process according to claim 7wherein said alcohol is methanol.
 12. The process according to claim 7or 11 employing as said yeast Pichia pastoris NRRL Y-11430.
 13. Theprocess according to claim 7 or 11 employing as said yeast Pichiapastoris NRRL Y-11431.
 14. The process according to claim 7 or 11employing as said yeast Hansenula polymorpha NRRL Y-11432.
 15. Theprocess according to claim 7 further comprising the steps of:(a)removing a stream of said aqueous ferment from said fermentation means,(b) separating said removed stream of aqueous ferment into a stream ofseparated cells, and a stream comprising separated aqueous mediacontaining residual dissolved minerals, (c) water-washing said separatedcells and thereby substantially separating said traces of mineral saltsfrom the cells, leaving a wet cell mass, (d) drying said wet cell massto produce dried cells, and (e) recycling the water-washings andseparated aqueous media to said culturing to provide therein at least inpart said makeup water and said mineral salts.
 16. The process accordingto claim 7 further comprising the steps of:removing a portion of saidaqueous ferment as fermenter effluent containing said cellular phase,and aqueous extracellular phase including residual mineral salts, anddrying said aqueous ferment effluent containing said cellular phase andsaid aqueous phase, thereby producing a dried cellular productcontaining residual water-soluble substances including salts.
 17. Theprocess of claim 7 further comprising the steps of:(a) removing a streamof said aqueous ferment effluent from said fermentation means, (b)centrifuging said removed aqueous ferment, thereby producing a stream ofconcentrated cells, and a stream of lean recycle mineral salts aqueousliquor, (c) water-washing said concentrated cells to produce a stream ofwater-washings containing further residual mineral salts, and a streamof washed cells, (d) drying said washed cells, and (e) recycling atleast a part of at least one of said lean recycle liquor and saidwater-washings to said aqueous ferment to provide therein at least aportion of said makeup water and mineral salts.
 18. The processaccording to claim 5 further employing in said aqueous ferment at leastone element selected from the group consisting of sodium, cobalt,molybdenum, boron, and selenium.
 19. The process according to claim 18further employing in said aqueous ferment at least one vitamin.
 20. Theprocess according to claim 19 wherein said vitamin is at least one ofbiotin or thiamine.
 21. The process according to claim 20 wherein saidmineral salts comprise a primary mineral salts medium and a tracemineral salts medium, and wherein said primary mineral salts mediumsupplies said P, K, Mg, S, and Ca; and said trace mineral salts mediumfurnishes said Fe, Zn, Mn, and Cu.
 22. The process according to claim 21wherein said carbon energy substrate is methanol or ethanol.
 23. Theprocess according to claim 22 further comprising the steps of:admixingsaid vitamin with an aqueous solution of said methanol or ethanol,adding said trace mineral salts medium thereto; and feeding theresulting admixture into said aqueous ferment separately from saidprimary mineral salts medium.
 24. The process according to claim 1wherein said carbon energy substrate is selected from the groupconsisting of carbohydrates, alkenes, alcohols, ketones, aldehydes,acids, and esters, of 1 to 20 carbon atoms per molecule; α-olefins ofC₁₄ to C₁₈ ; and normal paraffins of 10 to 20 carbon atoms per molecule.25. The process according to claim 24 wherein said carbon energysubstrate is said carbohydrate, and is selected from the groupconsisting of glucose, fructose, galactose, lactose, sucrose, starch,dextrin, and mixtures.
 26. The process according to claim 24 whereinsaid carbon energy substrate is said normal paraffin, and is selectedfrom the group consisting of decane, undecane, dodecane, tridecane,tetradecane, pentadecane, hexadecane, octadecane, eicosane, andmixtures.
 27. The process according to claim 24 wherein said carbonenergy substrate is said alcohol, ketone, aldehyde, acid, or ester, andis selected from the group consisting of methanol, ethanol, ethyleneglycol, propylene glycol, 1-propanol, 2-propanol, glycerol, 1-butanol,2-butanol, 3-methyl-1-butanol, 1-pentanol, 2-hexanol, 1,7-heptanediol,1-octanol, 2-decanol, 1-hexadecanol, 1-eicosanol, acetone, 2-butanone,4-methyl-2-pentanone, 2-decanone, 3-pentadecanone, 2-eicosanone,formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, hexanal,7-methyloctanal, tetradecanal, eicosanal, acetic acid, propionic acid,butyric acid, glutaric acid, 5-methylhexanoic acid, azelaic acid,dodecanoic acid, eicosanoic acid, methyl formate, methyl acetate, ethylacetate, propyl butyrate, isopropyl hexanoate, hexyl 5-methyloctanoate,octyl dodecanoate, and mixtures.
 28. In a fermentation process ofproducing yeast cells wherein at least one yeast is cultured infermentation means characterized as being free from physical means toremove liquid medium from said aqueous ferment without removing cells,under aqueous aerobic fermentation conditions in an aqueous fermentcomprising yeast cells and aqueous liquor employing yeast-assimilablecarbon energy substrate, assimilable nitrogen source, water, oxygen, andmineral salts,removing aqueous ferment as effluent from said fermentermeans, and recovering the so-produced yeast cells as a single cellprotein product, wherein said carbon energy substrate is selected fromthe group consisting of carbohydrates, alcohols, ketones, aldehydes,acids, and esters, of 1 to 20 carbon atoms per molecule; and normalparaffins of 10 to 20 carbon atoms per molecule, wherein the improvementcomprises: employing as said yeast a species selected from the groupconsisting of Pichia pastoris NRRL Y-11430, Pichia pastoris NRRLY-11431, and Hansenula polymorpha NRRL Y-11432; and feeding sufficientsaid mineral salts to said aqueous ferment at a rate effective tomaintain in said aqueous ferment the following elements in at least thedesignated minimum amounts, per liter of aqueous ferment: P 1.9 g, K 1g, Mg 0.15 g, Ca 0.06 g, S 0.1 g, Fe 6 mg, Zn 2 mg, Cu 0.6 mg, and Mn0.6 mg, thereby maintaining a cell density in said aqueous ferment ofabout 60 to 160 grams, on a dried basis, per liter of aqueous ferment.29. The process according to claim 28 wherein said carbon energysubstrate is said carbohydrate and is selected from the group consistingof glucose, fructose, galactose, lactose, sucrose, starch, dextrin, andmixtures.
 30. The process according to claim 28 wherein said carbonenergy substrate is said normal paraffin, and is selected from the groupconsisting of decane, undecane, dodecane, tridecane, tetradecane,pentadecane, hexadecane, octadecane, eicosane, and mixtures.
 31. Theprocess according to claim 28 wherein said carbon energy substrate issaid alcohol, ketone, aldehyde, acid, or ester, and is selected from thegroup consisting of methanol, ethanol, ethylene glycol, propyleneglycol, 1-propanol, 2-propanol, glycerol, 1-butanol, 2-butanol,3-methyl-1-butanol, 1-pentanol, 2-hexanol, 1,7-heptanediol, 1-octanol,2-decanol, 1-hexadecanol, 1-eicosanol, acetone, 2-butanone,4-methyl-2-pentanone, 2-decanone, 3-pentadecanone, 2-eicosanone,formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, hexanal,7-methyloctanal, tetradecanal, eicosanal, acetic acid, propionic acid,butyric acid, glutaric acid, 5-methylhexanoic acid, azelaic acid,dodecanoic acid, eicosanoic acid, methyl formate, methyl acetate, ethylacetate, propyl butyrate, isopropyl hexanoate, hexyl 5-methyloctanoate,octyl dodecanoate, and mixtures.
 32. The process according to claim 28wherein said carbon energy substrate is an alcohol of 1 to 4 carbonatoms.
 33. The process according to claim 32 maintaining in each literof said aqueous ferment: P 1.9 to 20 g, K 1 to 20 g, Mg 0.15 to 3 g, Ca0.06 to 1.6 g, S 0.1 to 8 g, Fe 6 to 140 mg, Zn 2 to 100 mg, Cu 0.6 to16 mg, and Mn 0.6 to 20 mg.
 34. The process according to claim 33maintaining in each liter of said aqueous ferment: P 2.2 to 10 g, K 1.5to 10 g, Mg 0.3 to 1.2 g, Ca 0.08 to 0.8 g, S 0.2 to 5 g, Fe 9 to 80 mg,Zn 3 to 40 mg, Cu 1 to 10 mg, and Mn 0.9 to 8 mg.
 35. The processaccording to claim 34 wherein said alcohol is methanol or ethanol. 36.The process according to claim 35 further employing in said aqueousferment at least one of the group consisting of sodium, cobalt,molybdenum, boron, and selenium.
 37. The process according to claim 35further employing in said aqueous ferment at least one vitamin.
 38. Theprocess according to claim 37 wherein said vitamin is biotin, thiamine,or both.
 39. The process according to claim 38 wherein said mineralsalts comprises a primary mineral salts medium and a trace mineral saltsmedium, and wherein said primary mineral salts medium supplies said P,K, Mg, S, and Ca, and said trace mineral salts medium furnishes said Fe,Zn, Mn, and Cu; and said process further comprises the steps of:admixingsaid vitamin with an aqueous solution of said methanol or ethanol,adding the trace mineral salts medium thereto, and feeding the resultingtrace mineral salts medium/vitamin admixture into said aqueous fermentseparately from said primary mineral salts medium.
 40. The processaccording to claim 38 wherein said aqueous fermentation conditionsinclude fermentation temperature in the range of 25° C. to 65° C., pH inthe range of about 3 to 7, pressure in the range of about 0 to 150 psig,and fermentation time in the range of about 2 to 30 hours based onaverage retention.
 41. The process according to claim 40 wherein saidalcohol is methanol.
 42. The process according to claim 41 furthercomprising the steps of:water-washing said aqueous ferment effluent byadmixture thereof with water, separating aqueous liquor includingmineral salts remaining in the separated media from the cells, leaving awet cell mass, drying the wet cell mass to produce dried cells, andrecycling the water-washings and separated mineral salts to said aqueousferment to provide therein at least in part said makeup water and saidmineral salts.
 43. The process according to claim 41 further comprisingthe step of drying the aqueous ferment effluent from said culturingincluding cells and residual mineral salts, thereby producing a driedcell product containing residual water-soluble substances includingmineral salts.
 44. The process of claim 41 further comprising the stepsof:centrifuging said removed aqueous ferment effluent, thereby producinga stream of concentrated cells, and a stream of lean recycle liquor,water-washing said concentrated cells, separating water-washings fromsaid cells to produce a stream of water-washings containing residualmineral salts, and a stream of washed cells, drying said washed cells,and recycling at least in part at least one of said lean recycle liquorand said water-washings to said aqueous ferment to provide therein atleast a portion of said makeup water and said mineral salts.
 45. Theprocess according to claim 35 maintaining a cell density of about 70 to150 grams, on a dried basis, per liter of aqueous ferment.
 46. Theprocess according to claim 35 maintaining a cellular yield of about 30to 110 grams per 100 grams substrate charged.
 47. A method of producinga protein material which comprises culturing a biologically pure yeastselected from the group consisting of Pichia pastoris NRRL Y-11430,Pichia pastoris NRRL Y-11431, or Hansenula polymorpha NRRL Y-11432, inan aqueous medium, employing an oxygenated hydrocarbon as carbon energysubstrate, aerobic aqueous fermentation conditions, mineral salts,assimilable nitrogen source, and oxygen, and recovering from theresulting single cell microorganisms a protein material.
 48. The processof claim 47 wherein said yeast is said Pichia pastoris NRRL Y-11430. 49.The process according to any of claims 48, 66, or 67 wherein saidalcohol contains 1 to 4 carbon atoms per molecule.
 50. The processaccording to claim 49 wherein said alcohol is methanol.
 51. The processaccording to claim 47 wherein said yeast is said Pichia pastoris NRRLY-11431.
 52. The process according to claim 47 wherein said yeast issaid Hansenula polymorpha NRRL Y-11432.
 53. The process according toclaim 47 wherein said carbon energy substrate is selected from the groupconsisting of alcohols, ketones, esters, ethers, acids, and aldehydes,which are substantially water-soluble and contain up to about 10 carbonatoms per molecule.
 54. In a continuous process of biochemicalconversion wherein a carbon energy substrate is converted to products offermentation, which comprises culturing at least one yeast under aerobicaqueous fermentation conditions, in aqueous ferment comprising acellular phase and an aqueous extracellular phase, employing ayeast-assimilable carbon energy substrate, assimilable nitrogen source,water, molecular oxygen, and mineral salts,wherein the improvementcomprises: employing a yeast selected from the group consisting ofPichia pastoris NRRL Y-11430, Pichia pastoris NRRL Y-11431, andHansenula polymorpha NRRL Y-11432, and feeding said mineral salts tosaid aqueous ferment in an amount sufficient to maintain in said aqueousferment the following elements in at least the designated amounts perliter of aqueous ferment: P 1.9 g, K 1 g, Mg 0.15 g, Ca 0.06 g, S 0.1 g,Fe 6 mg, Zn 2 mg, Cu 0.6 mg, and Mn 0.6 mg, and said carbon energysubstrate in amounts required for maximum growth of cells, maintaining ayeast cell density in said aqueous ferment of about 60 to 160 grams on adried basis per liter of said aqueous ferment, and producing at leastone product of fermentation.
 55. The process of claim 54 wherein saidcarbon energy substrate is methanol.
 56. The process according to claim55 wherein said biochemical conversion employs as said carbon energysubstrate methanol, ethanol, or glucose, and produces fermentationproducts comprising carbon dioxide and single cell protein.
 57. Afermentation process of producing yeast protein product, which comprisesculturing a yeast under aqueous aerobic fermentation conditions, inaqueous ferment comprising yeast cells and aqueous liquor, employingyeast-assimilable carbon energy substrate, assimilable nitrogen source,water, oxygen, and mineral salts comprising a primary salts medium and atrace mineral salts,feeding sufficient said mineral salts to saidaqueous ferment to maintain the following elements in at least theminimum amount, per liter of aqueous ferment: P 1.9 g, K 1 g, Mg 0.15 g,Ca 0.06 g, S 0.1 g, Fe 6 mg, Zn 2 mg, Cu 0.6 mg, and Mn 0.6 mg, therebymaintaining a cell density in said aqueous ferment of about 60 to 160grams, on a dried basis, per liter of aqueous ferment, employing afermenter characterized as being free from physical means to removeliquid medium from said aqueous ferment without removing cells, andrecovering the resulting yeast microorganisms, wherein said carbonenergy substrate is selected from the group consisting of carbohydrates,alcohols, ketones, aldehydes, acids, and esters, of 1 to 20 carbon atomsper molecule; and normal paraffins of 10 to 20 carbon atoms permolecule.
 58. The process according to claim 57 wherein said yeast isselected from the group consisting of Pichia pastoris NRRL Y-11430,Pichia pastoris NRRL Y-11431, and Hansenula polymorpha NRRL Y-11432, andsaid carbon energy substrate is methanol or ethanol.